Sterilizing Equipment

[deleted-331039]

Hi,

I will be growing the bacteria found in yogurt, and I'm wondering how I can sterilize the filter disks I'm using to test antimicrobial effectiveness without using an autoclave. For the metal utensils, I'm planning to simply clean them with rubbing alcohol, but I'm not sure if that could be done with filter paper. Please note that I do not have access to a fancy lab at the moment.

I'm also wondering if the bacteria grown directly from yogurt would grow in a uniform fashion and not in several small colonies? Here is the link to the method I'm using:
https://www.sciencebuddies.org/science- ... p072.shtml

Thanks!
Mad_Scientist

[deleted-332001]

Hi Mad_Scientist! The easiest way to solve your problem would be to buy sterile filter disks. Otherwise, you could soak with 70% isopropyl alcohol or 70% ethanol and then let it dry. However, the problem with this approach is that you need a sterile environment for the disks to dry. Otherwise the disks could get contaminated with bacteria by the time they dry. So, if you decide to take this approach, you should wipe everything in the vicinity of the filter disks with 70% alcohol as well.

To answer your second question, the density of colonies depends on the concentration of bacteria. If the bacteria is present at a low concentration in the yogurt, then you may see a few colonies that grow in size over time. Each colony is produced by a single bacterium. If the yogurt has a high concentration of bacteria, you may see a film of bacteria.

Best regards,
Jen

[deleted-331039]

Thanks for the quick reply!

I just did some research and apparently yogurt contains from 3 billion cfu/ml to 10 billion cfu/ml. Would this be considered enough bacteria to grow a nice film? If not, is there anyway to make the bacteria grow in a film? This would be important since I'm using the diffusion method to determine disinfectant ability.

Thanks!
Mad_Scientist

[deleted-284605]

Hi Mad_Scientist,

I think as long as you coat the entire plate with at least a bit of yogurt, you'll get a so-called "lawn" of bacteria (continuous growth in two dimensions rather than many small colonies).

If you can't find sterile disks, you could also try microwaving them for 30-60 seconds (be careful though- I'm not sure how flammable they might be)!

Good luck,
Megan

[deleted-331039]

Thanks! I'll consider using either the ethanol or microwave method. If I do use the microwave method, should I soak the disks in water first or should I wrap them in aluminum foil? (I'm kind of worried that the disks will burn) I'm also wondering how far apart I should place each filter disk from each other. I'm thinking about placing them about 3 cm away from each other but I'm not exactly sure since I couldn't find an exact procedure for that.

Another quick question: should I dilute the yogurt before spreading them on the dishes? The initial procedure said to make a 50:50 dilution.

Thanks for the help!
Mad_Scientist

[deleted-284605]

Don't put foil in the microwave!!! That will start a fire!

Wetting the disks and nuking for 30 seconds should be fine, though whatever you buy will probably be sterile anyway! Dousing with ethanol and then allowing the disks to dry under a bowl or some other clean space should be fine too.

What sort of anti-microbial agents are you using? Placing ~3 disks/plate in a triangle shape (to maximize distance between them) will probably be okay, but the safest bet would be to put one disk in the center of each plate. Just depends on how many plates you have.

The same goes for the yogurt dilution. I would test a range of dilutions (e.g. undiluted, 75%, 50%, and 25% yogurt) to see which gives the best bacterial lawn before adding the disks. If you don't have enough plates to do all this, I'd just try the suggested 50% yogurt mix. These experiments should have been tested, so that ratio was suggested for a reason!

[deleted-331039]

Thanks for the advice! I sterilized the filter disks today! Now I'm just wondering, if I were to sterilize glass and metal equipment, should I boil them, bake them, or just simply wipe them down with some alcohol? I want to get rid of as much bacteria, and I'm not sure which method is most effective.

By the way, I'm going to go with the 75% yogurt dilution.

Mad_Scientist

[deleted-288920]

Hi there!

If I were you, I would wipe them down with alcohol. I work in a microbiology lab and wiping with alcohol is exactly how I clean my spatulas.

Hope that helps!
Nikki

[deleted-331039]

I'm just wondering if there is a way to speed up the growth of the yogurt bacteria because I just read that it takes about 6 days... I just tried incubating the plates at high temperatures, but since I used gelatine plates, the medium simply melted. Thanks for the help!

Mad_Scientist

[deleted-291782]

Hello,

For the growth of the bacteria, I would just be patient! Raising the temperature too far will likely kill the cells, and will melt the medium upon which they are growing. Other than giving them optimal temperature and a nutrient-rich medium, there isn't much you can do to accelerate their growth.

Best,
Pharma

[deleted-352197]

Uniform fashion, great.
All the best
Go for it.